Despite the dramatic improvement in HIV-associated morbidity and mortality from combination antiretroviral therapy, a number of challenges remains including the long-term persistence of multiple, latent viral reservoirs capable of reactivation in the absence of ART. Any effort to eradicate these reservoirs as part of a cure initiative requires understanding of the dynamics and control of HIV reactivation and replication in tissues and cells harboring the virus long-term. The proposed studies analyze the genitourinary (GU) tract as a unique compartment and reservoir for HIV. Our prior work has demonstrated that renal epithelial cells are a unique viral compartment and in preliminary data we show that viral RNA sequences derived from the urine of males and females form separate cluster(s) from those derived from PBMC and plasma, consistent with viral compartmentalization in the GU tract. Viral detection in the urine allows for repeated sampling of this compartment, which can be particularly useful in viral rebound studies. To determine the source of urine- derived sequences in males, cell free and cell-associated viral sequences will be isolated and genetically characterized simultaneously from urine and semen. To assess the contribution of the GU tract to virus rebound, simultaneous and longitudinal phylogenetic sequence analysis will be performed on urine-derived HIV nucleic acids in comparison with PBMC and plasma, during ART and after treatment interruption in patients enrolled in the ACTG 5345 trial (Specific Aim 1). To assess the long-term in vivo persistence of HIV nucleic acids in the upper GU tract, DNA and RNA amplification, in situ hybridization and deep sequencing will be performed on kidney biopsies from HIV positive subjects that have prolonged suppression on ART. Deep sequencing will detect preferential HIV integration sites associated with clonal expansion of infected renal epithelial cells (Specific Aim 2). Specific Aim 3 will define the long-term potential of the kidney as a latent and reactivatable reservoir for HIV. A dual fluorescent viral construct, that allows identification and purification of cell populations containing transcriptionally active or silenced HIV, will be used to define physiologic and pharmacologic strategies for virus reactivation (from latency) as well as the fate of RTE upon activation. In an ex vivo approach, RTE cells isolated and cultured from the urine of suppressed HIV-1 positive subjects will be assessed using similar activation strategies and compared to stimulation of simultaneously derived PBMC. Collectively, this work will further characterize the GU tract associated virus as well as define characteristics of latency and transcriptional activation in renal epithelium, a unique viral compartment that may contribute to long-term HIV persistence.